HIF/hypoxia-inducible factor hydroxylase

Nuclear HIF-1 stabilizes in response to TRC160334 (100~400 μM; 4 hours; Hep3B cells) in a dose-dependent manner [1]. Hep3B cells treated with TRC160334 (75~300 μM; 4 hours) exhibit dose-dependent transcriptional activation of HIF-1. HIF target genes, including adrenomedullin and EPO, express themselves in TRC160334 in a dose-dependent manner [1].

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TRC160334 Treatment Results in Activation of HIF [1]
TRC160334 treatment resulted in a dose-dependent stabilization of HIF-1α in Hep3B cells (fig. 2a) leading to dose-dependent activation of HIF (fig. 2b). As a result, Hep3B cells treated with TRC160334 showed a dose-dependent expression of HIF target genes such as EPO (EC50 84 µM) and adrenomedullin (EC50 91 µM) (fig. 2c, d).

TRC160334 (0.1 and 0.3 mg/kg; i.p.) significantly lowers serum creatinine and blood urea nitrogen [1]. TRC160334 (0.3 and 0.6 mg/kg; i.p.) demonstrated a trend toward decreasing acute tubular necrosis [1]. TRC160334 considerably lowers the dose-dependent increase in electrolyte excretion. Pre-ischemia treatment with TRC160334 resulted in considerable induction of HSP70 in the kidney for 6 hours, whereas post-ischemia therapy with TRC160334 resulted in significant induction of HSP70 in the kidney for 12 hours compared to the matching vehicle control [1].

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Pre- and Postischemia Treatment by TRC160334 Improves Renal Function in Ischemic AKI [1]
Pre- as well as postischemia treatment with TRC160334 results in a remarkable improvement in renal function. For all parameters studied improvement with TRC160334 treatment, unless mentioned, is reported at its peak effect level, i.e. either day 1 and/or day 2. A significant increase in serum creatinine (∼7- to 8-fold, peak rise) from baseline levels was observed in untreated rats (vehicle control) 24 h after the induction of renal ischemia/reperfusion (I/R; 35 min bilateral renal artery occlusion). Serum creatinine levels did not rise in the TRC160334 treatment group as much as it did in the control group, preischemia treatment with TRC160334 dose-dependently reduced the elevation of serum creatinine. At the highest dose (TRC-0.3 mg-pre), TRC160334 significantly reduced serum creatinine by 42% (p < 0.01) on day 1 and 66% (p < 0.05) on day 2 as compared to vehicle control rats (fig. 3a).

Similarly, postischemic treatment with TRC160334 initiated at 2 h after the onset of renal ischemia significantly reduced serum creatinine levels. At the highest dose (TRC-0.6 mg-post), TRC160334 significantly reduced serum creatinine by 23% (p < 0.01) on day 1 and 71% (p < 0.01) on day 2 as compared to vehicle control rats (fig. 3b).

Preischemic treatment with TRC160334 dose-dependently reduced elevated BUN levels. At the highest dose (TRC-0.3 mg-pre), TRC160334 significantly reduced BUN by 24% (p < 0.05) on day 1 and 55% (p < 0.05) on day 2 as compared to vehicle control rats (fig. 3c).

Also, in case of postischemic treatment, TRC160334 dose-dependently reduced elevated BUN levels. At the highest dose (TRC-0.6 mg-post), TRC160334 significantly reduced BUN by 16% (p < 0.01) on day 1 and 48% (p < 0.01) on day 2 as compared to vehicle control rats (fig. 3d).

Ischemic AKI is characterized by the presence of an oliguric phase and a postischemic diuretic phase. Untreated rats show both these features with oligurea phase up to 24 h and diuretic thereafter up to 48 h. TRC160334 administered under both conditions, preischemia as well as postischemia, abolished such effect on urine output dose-dependently and at the highest dose, urine output was significantly maintained to preischemia level in TRC160334-treated rats (fig. 4a, b).

Renal ischemia causes significant increase in fractional excretion of electrolytes such as fractional excretion of sodium (FeNa). Peak rise in FeNa was observed at 24 h postischemia onset. Preischemic treatment with TRC160334 significantly reduced the rise in electrolyte excretion dose dependently. On day 1, 80% (p < 0.05) reduction and on day 2, 72% (p < 0.05) reduction in FeNa was observed at the highest dose (TRC-0.3 mg-pre) treated rats (fig. 4c).

Similarly, postischemic treatment with TRC160334 also reduced the rise in electrolyte excretion. On day 1, 59% (p < 0.05) reduction and on day 2, 79% (p < 0.05) reduction in FeNa was observed at the highest dose (TRC-0.6 mg-post) treated rats (fig. 4d).

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Pre- and Postischemia Treatment by TRC160334 Results in Preservation of Renal Architecture [1]
Histopathological examination of representative HE sections of kidneys of vehicle control animals after ischemia reperfusion injury showed a moderate degree of acute tubular necrosis (ATN); characterized by cystic dilatation of tubules, dilated tubules with loss of brush border, tubular cells necrosis, and widening of interstitial space with cellular infiltrates (fig. 5a). Scoring for ATN 7 days after reperfusion revealed a better preserved renal morphology after preischemic treatment with TRC160334 (TRC-0.3 mg-pre). Preischemic treatment with TRC160334 showed significant improvement in the renal histology. Most of the renal tubules and tubulointerstitial space showed a milder degree of ATN (fig. 5b, c). A trend towards preserved morphology after postischemic treatment with TRC160334 (TRC-0.6 mg-post) compared to the respective vehicle was observed. Postischemic treatment with TRC160334 also showed reducing trends for ATN (fig. 5d, e).

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Pre- and Postischemia Treatment by TRC160334 Results in HSP70 Induction [1]
Expression of EPO, adrenomedullin and HSP70 were monitored in kidneys of vehicle-treated and TRC160334-treated animals by immunoblotting. Expression of EPO and adrenomedullin was found to be unaffected under experimental conditions. Preischemic treatment with TRC160334 resulted in a pronounced induction of HSP70 in kidneys by 6 h (fig. 6a) while postischemic treatment with TRC160334 resulted in a pronounced induction of HSP70 in kidneys by 12 h as compared with the respective vehicle control (fig. 6b).

Western Blot Analysis[1]
Cell Types: Hep3B cells
Tested Concentrations: 100~400 μM
Incubation Duration: 4 hrs (hours)
Experimental Results: Result in dose-dependent stabilization of nuclear HIF-1.

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HIF-α Stabilization [1]
Hep3B cells were treated with vehicle or TRC-160334 for indicated doses for 4 h. Nuclear extracts were then prepared and nuclear proteins were separated on SDS-PAGE followed by immunoblotting. Immunoblots were probed with HIF-1α antibody.

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HIF Transactivation [1]
Hep3B cells were transiently transfected with HIF-1 luciferase vector (pHIF1-Luc) along with normalization vector, β-galactosidase. Transfected cells were treated with vehicle or TRC-160334 for indicated doses for 4 h. Cell lysates were then prepared and analyzed for luciferase and β-galactosidase activity. Results were expressed as fold induction of HIF activation over that for vehicle control.

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Expression of EPO [1]
Hep3B cells were treated with vehicle or TRC-160334 for indicated doses for 16 h. At the end of 16 h, cell culture medium was collected and centrifuged to remove any debris. Supernatant obtained was analyzed for EPO by ELISA. The results were expressed as fold induction as compared to vehicle control.

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Expression of Adrenomedullin [1]
Hep3B cells were treated with vehicle or TRC-160334 for indicated doses for 6 h. At the end of 6 h, cells were lysed and total RNA was isolated. Expression of adrenomedullin mRNA along with expression of 18S rRNA was monitored by real-time PCR employing ABI 7900 HT. Adrenomedullin mRNA expression was normalized relative to the expression of 18S rRNA. The results were expressed as fold induction of adrenomedullin mRNA relative to vehicle control.

Animal/Disease Models: SD (SD (Sprague-Dawley)) male rats (250–300 g) [1]
Doses: 0.1 and 0.3 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: Serum creatinine and blood urea nitrogen were Dramatically diminished.

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Animal/Disease Models: SD (SD (Sprague-Dawley)) male rats (250–300 g) [1]
Doses: 0.3 and 0.6 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: demonstrated a trend towards diminished acute tubular necrosis.

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Animals [1]
Sprague-Dawley male rats (250–300 g body weight) were derived from an in-house bred colony. These rats were housed in a 12-hour light-dark cycle in a specific pathogen-free facility with controlled temperature and humidity, and allowed free access to food and water.

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Experimental Protocols and Groups [1]
Test Compound and Formulation [1]
For testing the in vivo efficacy of TRC-160334, it was formulated into an injectable solution using clinically accepted formulation excipients. Irrespective of the dose 1 ml/kg of the test compound formulation was injected intraperitoneally. Phosphate-buffered saline pH adjusted to 7.4 was used as vehicle.

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Induction of Renal Ischemia/Reperfusion [1]
Rats were anaesthetized with pentobarbitone sodium (50 mg/kg, 2 ml/kg, i.p). After ensuring proper induction of anaesthesia, abdominal fur was removed by means of a hair clipper and the animal was kept on a homeothermic blanket for maintaining body temperature at 37 ± 0.5°C throughout the surgical procedure. Bilateral renal ischemia was induced according to a previously described procedure. Briefly, a midline incision was made in the abdominal region and the viscera was removed and kept on a side in gauze moistened with prewarmed saline (37°C). Both renal artery and vein were isolated and occluded by means of a microvessel clip for 35 min. After occlusion of renal artery and vein, abdomen was sutured with a 3-0 suture in order to prevent heat loss. At the time of reperfusion (35 min after occlusion), the abdomen was once again opened, and proper occlusion of both kidneys was visually ensured (marked cyanosis). Reperfusion of kidneys was done by removing microvessel clips. 0.5 ml of warm saline (37°C) was instilled on each kidney, after which the kidneys were observed visually for proper reperfusion (returning back to the normal color). The animal was sutured again and kept in an incubator (maintained at 37°C) for recovery from anesthesia. Each animal then received benzylpenicillin (33,333 IU, 0.1 ml/kg s.c., b.i.d. for 2 days) and bupronorphine (0.15 mg, 0.5 ml/kg, i.m., b.i.d. for 2 days) as postoperative care. Animals which fail led to show complete cyanosis just before reperfusion or complete reperfusion after the removal of microvessel clips of either of kidney were excluded from study.

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TRC-160334 Treatment [1]
Preischemia Treatment [1]
Animals were divided into groups (at least 9 per group) as follows:
Group 1 (Veh-pre): Vehicle control animals were injected with 1 ml/kg of vehicle (PBS, pH 7.4) before the induction of renal ischemia (control for groups 2 and 3).
Group 2 (TRC-0.1 mg-pre): Animals pretreated with three injections of TRC-160334 (0.1 mg/kg) before the induction of renal ischemia.
Group 3 (TRC-0.3 mg-pre): Animals pretreated with three injections of TRC-160334 (0.3 mg/kg) before the induction of renal ischemia.
Animals from groups 2 and 3 received TRC-160334 injections by the intraperitoneal route. First injection at 12 h, second at 6 h and a third injection at 1 h before surgical induction of renal ischemia. Animals from group 1 received the vehicle in a similar fashion.

Postischemia Treatment [1]
Animals were divided into groups (at least 9 per group) as follows:
Group 4 (Veh-post): Vehicle control animals were injected with 1 ml/kg of vehicle (PBS, pH 7.4) after the induction of renal ischemia (control for groups 5 and 6).
Group 5 (TRC-0.3 mg-post): Animals received TRC-160334 injections (3 per group) starting from 2 h after the onset of renal ischemia.
Group 6 (TRC-0.6 mg-post): Animals received TRC-160334 injections (3 per group) starting from 2 h after the onset of renal ischemia. Animals from groups 5 and 6 received TRC-160334 injections by the intraperitoneal route starting from 2 h (0.3 mg/kg to group 5 and 0.6 mg/kg to group 6 animals), with a second and a third injection at 6 and 10 h (0.1 mg/kg to group 5 and 0.2 mg/kg to group 6 animals) after the surgical induction of renal ischemia. Animals from group 4 received vehicle in a similar fashion.

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Serum and Urine Biochemistry [1]
Timed blood samples from each animal were collected by sublingual puncture and serum (150 µl) was separated. Blood sampling was done at various time points starting from 24 h before and up to 72 h after the induction of renal ischemia. Similarly pooled urine samples were collected for each animal by keeping animals in metabolic cages. All animals were acclimatized to metabolic cages 1 day before the actual collection. Total urine volume at each time point for each animal was recorded. Thereafter, on day 7 after the onset of ischemia, at least 4 animals per group were killed and both kidneys were processed for morphological studies. Serum and urine samples were analyzed for creatinine and sodium, while BUN was estimated only in serum. All biochemical parameters were estimated using a fully automated clinical chemistry analyzer Olympus AU400. Figure 1 provides a scheme of the experimental protocol used for the evaluation of the efficacy of TRC-160334 by pre- or postischemia treatment.

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Gene Expression Studies [1]
Kidneys of vehicle and TRC-160334 treated animals were processed for whole cell extract preparation. Proteins were separated on SDS-PAGE followed by immunoblotting, employing EPO antibody, adrenomedullin antibody, and HSP70 antibody.

[1]. Treatment with a novel hypoxia-inducible factor hydroxylase inhibitor (TRC160334) ameliorates ischemic acute kidney injury. Am J Nephrol. 2012;36(3):208-218.

Background: Hypoxia-inducible factor (HIF) transcriptional system plays a central role in cellular adaptation to low oxygen levels. Preconditional activation of HIF and/or expression of its individual target gene products leading to cytoprotection have been well established in hypoxic/ischemic renal injury. Increasing evidence indicate HIF activation is involved in hypoxic/ischemic postconditioning of heart, brain and kidney. Very few studies evaluated the potential benefits of postischemia HIF activation in renal injury employing a pharmacological agent. We hypothesized that postischemia augmentation of HIF activation with a pharmacological agent would protect renal ischemia/reperfusion injury. For this, TRC160334, a novel HIF hydroxylase inhibitor, was used.
Methods: TRC160334, a novel HIF hydroxylase inhibitor, was synthesized. Ability of TRC160334 for stabilization of HIF-α and consequent HIF activation was evaluated in Hep3B cells. Efficacy of TRC160334 was evaluated in a rat model of ischemia/reperfusion-induced AKI. Two different treatment protocols were employed, one involved treatment with TRC160334 before onset of ischemia, the other involved treatment after the reperfusion of kidneys.
Results: TRC160334 treatment results in stabilization of HIF-α leading to HIF activation in Hep3B cells. Significant reduction in renal injury was observed by both treatment protocols and remarkable reduction in serum creatinine (23 and 71% at 24 and 48 h, respectively, p < 0.01) was observed with TRC160334 treatment applied after reperfusion. Urine output was significantly improved up to 24 h by both treatment protocols.
Conclusion: The data presented here provide pharmacologic evidence for postischemia augmentation of HIF activation by TRC160334 as a promising and clinically feasible strategy for the treatment of renal ischemia/reperfusion injury.[1]

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Our findings with TRC160334, a novel HIF hydroxylase inhibitor, are in agreement with earlier reports, wherein pharmacological inhibition of HIF hydroxylases during preischemia has shown renal protection. In addition, our findings are first to demonstrate efficacy of HIF hydroxylase inhibitor in ameliorating ischemic AKI, when administered during postischemia. These findings highlight the potential of TRC160334, for its clinical application for prevention and for treatment of ischemic AKI. [1]

C14H13N3NA2O5S

381.31

1293290-41-1

1293290-41-1 (sodium);1293290-44-4 (potassium);1293289-69-6 (free acid); 1293290-45-5 (calcium);

Typically exists as solids at room temperature

O=C([O-])CNC(C1=C([O-])N=C(SC2=C3CCCCC2)N3C1=O)=O.[Na+].[Na+]

KDOUTFVJRKEFFZ-UHFFFAOYSA-L

InChI=1S/C14H15N3O5S.2Na/c18-9(19)6-15-11(20)10-12(21)16-14-17(13(10)22)7-4-2-1-3-5-8(7)23-14;;/h21H,1-6H2,(H,15,20)(H,18,19);;/q;2*+1/p-2

sodium (2-oxido-4-oxo-7,8,9,10-tetrahydro-4H,6H-cyclohepta[4,5]thiazolo[3,2-a]pyrimidine-3-carbonyl)glycinate

TRC-160334 sodium salt; TRC 160334 di-sodium salt; TRC160334

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)